# 710: THERMOANAEROBACTER (KoKo) MEDIUM


## Main sol. 710

1	g	Tryptone (BD Bacto)	
1	g	Peptone (meat)	
1	g	Yeast extract (BD Bacto)	
1.6	g	K2HPO4	
1	g	NaH2PO4 x 2 H2O	
0.5	g	NH4Cl	
0.16	g	MgSO4 x 6 H2O	
1	ml	**Trace element solution SL-11**	
0.5	ml	Sodium resazurin (0.1% w/v)	
0.06	g	CaCl2 x 2 H2O	
1	g	NaHCO3	
5	g	D-Glucose	
1	ml	**Wolin's vitamin solution (10x)**	
0.3	g	L-Cysteine HCl x H2O	
0.3	g	Na2S x 9 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients (except calcium chloride, bicarbonate, glucose, vitamins, cysteine and sulfide), adjust pH to 7.0 and sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After sterilization add calcium chloride, glucose, cysteine and sulfide from sterile anoxic stock solutions autoclaved under 100% N2 gas atmosphere. Add bicarbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Vitamins are prepared under 100% N2 gas and sterilized by filtration. The pH of the complete medium should be at 7.0.
2. Note: Supplementation of medium with 10.00 g/l MOPS buffer (pH 6.9 - 7.0; 10% (w/v) anoxic stock solution) may enhance the buffer capacity of the medium.


## Trace element solution SL-11

5.2	g	Na2-EDTA x 2 H2O	
1.5	g	FeCl2 x 4 H2O	
70	mg	ZnCl2	
100	mg	MnCl2 x 4 H2O	
6	mg	H3BO3	
190	mg	CoCl2 x 6 H2O	
2	mg	CuCl2 x 2 H2O	
24	mg	NiCl2 x 6 H2O	
36	mg	Na2MoO4 x 2 H2O	
1000	ml	Distilled water	

1. Dissolve EDTA in 800 ml distilled water, adjust pH to 7 using 2 N NaOH and add ferrous chloride. After ferrous chloride has dissolved add other compounds. Finally adjust pH to 6.0 and bring volume to 1000 ml. 


## Wolin's vitamin solution (10x)

20	mg	Biotin	
20	mg	Folic acid	
100	mg	Pyridoxine hydrochloride	
50	mg	Thiamine HCl	
50	mg	Riboflavin	
50	mg	Nicotinic acid	
50	mg	Calcium D-(+)-pantothenate	
1	mg	Vitamin B12	
50	mg	p-Aminobenzoic acid	
50	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water